The long term objective of the proposed research is to understand the properties of retroviruses that determine the structures of their genomes, and to examine how structural and organizational properties of retroviral genomes affect replication success. Abnormal retroviral genomes that contain copies of cellular genes led to the discovery of oncogenes, and the ability of retroviruses to incorporate heterologous sequences contributes to retroviral pathogenesis. This proposal examines general and specific parameters that influence retroviral genome structure, using Moloney murine leukemia virus as a model system. The first specific aim is to determine what constraints limit retroviral RNA genome size and organization, and to examine the consequences of these limitations. The second specific aim is to pinpoint the sequences essential for the initiation of plus strand DNA synthesis in vivo, with a long-term goal of understanding how interactions between reverse transcriptase and viral nucleic acids influence the form of retroviral genomes. Artificially engineered retroviral RNAs that differ from wild type genomes in size or number will be constructed, and the ability of these genomes to be encapsidated and to generate retroviral DNA will be examined. The genetic consequences of deviation from standard genome organization will be examined in terms of replication fidelity, and a model for the generation of oncogenic retroviruses will be tested. The second specific aim will involve making targeted mutations in the polypurine tract region and then examining the mutations' effects on primer specificity and utilization in vivo, with a goal of determining whether the generation of the plus strand primer and its subsequent utilization involve the same sets of specific reverse transcriptase - nucleic acid interactions. A genetic selection for polypurine tract mutations will then be established. These studies have implications for two areas important to human health, as well as general benefits through expanding the understanding of retroviral biology. Understanding the effects of retroviral genomic alterations is important to the design of retroviral vectors and the evaluation of their safety. These studies may lead to the development of larger retroviral vectors that would facilitate simultaneous introduction of panels of genes or provide room for regulatory regions. Secondly, by examining mechanisms of genetic variation that retroviruses may employ to alter their pathogenic potential, these studies will provide information valuable to the development of anti-AIDS therapeutics.